Acetaminophen (APAP)-induced liver injury is a leading public health concern and the number one cause of acute liver failure in the United States. Existing pharmacologic strategies for the management of APAP overdose patients have evolved very little in the last several decades, and remain focused on reducing the toxicity of the reactive metabolite NAPQI, thereby reducing APAP hepatotoxicity. The hepatotoxic response to APAP is counterbalanced by a robust and timely regenerative response aimed at limiting progression of injury and stimulating hepatocyte proliferation. Because liver injury has already occurred at the time most patients are seen clinically, these patients would most likely benefit from therapies aimed at stimulating liver repair prior to the onset of complications such as infection and organ failure. However, such therapies are not currently available because the mechanisms that stimulate and regulate liver repair after APAP hepatotoxicity are poorly understood. Our strong preliminary studies indicate that the progression of APAP-induced liver injury and hepatocyte proliferation are controlled by the plasmin (ogen) system and its central regulator plasminogen activator inhibitor-1 (PAI-1). The central hypothesis framing these studies is that PAI-1 inhibits amplification of APAP hepatotoxicity by reducing plasmin (ogen)-driven proteolysis, and stimulates liver repair by its anti- protease and/or vitronectin-binding activitie. Our approach includes a combination of genetically-modified mice, recombinant human PAI-1 proteins with customized activities, novel small molecule PAI-1 inhibitors, and an FDA-approved inhibitor of plasminogen activation (tranexamic acid). The investigative team comprises experts in toxic liver injury and repair, PAI-1 biochemistry and function, and gene-regulation in acute and chronic liver disease. Specifically, we will: (Aim 1) Determine the mechanism whereby plasmin(ogen) activation exacerbates APAP-induced liver injury; (Aim 2) Determine the mechanism whereby PAI-1 stimulates liver repair after APAP toxicity; and (Aim 3) Determine the role of HIF-1a-regulated PAI-1 induction in liver injury and repair after APAP overdose. The insights gained will significantly advance the current understanding of liver repair after toxic lier injury, and highlight novel therapeutic strategies to prevent liver failure after APAP overdose, including potential repurposing of FDA-approved plasmin (ogen) inhibitors and mechanism-based translation of recombinant human PAI-1 proteins with customized activity to stimulate liver regeneration.